ALBERT

Description: Abstract 377 Hemophilia B represents a promising model for the development of adeno-associated viral (AAV) vectors-based gene therapeutics. In the first clinical trial for AAV serotype 2 mediated gene transfer of Factor IX (F.IX) to the liver of severe hemophilia B subjects, transgene expression was short-lived with a gradual decline of F.IX levels. The loss of transgene expression was accompanied by a transient transaminitis, which we hypothesized to be the result of the reactivation of a pool of capsid-specific memory CD8+ T cells originated from a previous exposure to wild-type AAV. These results were unanticipated since previous work in small and large animal models showed that AAV administration is uneventful, allowing prolonged expression of F.IX transgene at therapeutic levels. We developed an in vitro cytotoxicity assay using a human hepatocyte cell line expressing HLA-B*0702, a common MHC class I allele for which the AAV capsid immunodominant epitope VPQYGYLTL was identified. Using this model, we demonstrated that HLA-matched AAV-specific effector CD8+ T cells were able to lyse target hepatocytes transduced with AAV-2. We now use this in vitro model of CTL killing of AAV-transduced hepatocytes to demonstrate the efficacy of a novel strategy to circumvent undesirable immune response through the engagement of regulatory T cells. A recently characterized MHC Class II-restricted T cell epitope (Tregitope) in the Fc fragment of IgG has been shown to induce regulatory T cells in vitro and in vivo (Blood, 2008; 112: 3303-3311). AAV-specific HLA-B*0702 effector cells expanded in the presence of a human Tregitope peptide resulted in 79% to 89% inhibition of cytotoxic activity against peptide-pulsed and AAV-transduced target cells, respectively. These results were confirmed using PBMCs from 5 different donors. A similar degree of inhibition of CTL activity was observed for the HLA allele A*0101, which binds to the AAV-derived epitope SADNNNSEY; co-culture of effector cells with the Tregitope inhibited CTL-mediated killing by 60%. Interestingly, the same Tregitope efficiently mediated suppression of CTL activity in subjects carrying different HLA alleles, indicating a high level of promiscuity of Tregitope binding. Staining for the regulatory T cell markers CD4, CD25, and FoxP3 supported the hypothesis that Tregitopes suppress T cell responses by expanding regulatory T cells; 62.2% of the CD4+ population stained positive for CD25 and FoxP3 in PBMCs expanded against AAV epitopes in the presence of Tregitope, compared with PBMCs expanded against an AAV epitope alone (3.63%), or against an AAV epitope and an irrelevant control peptide (1.94%). Polyfunctional analysis for markers for T cell activation showed that CD8+ T cells incubated in the presence of Tregitope had an approximately 5-fold decrease in production of IL-2 and IFN-γand a 2-fold reduction in TNF-α production, indicating levels of activation close to naïve CD8+ T cells. We further characterized the mechanism of action of Tregitopes by showing that Tregitopes are required at the time of CD8+ T cell priming, as CTL activity of AAV-expanded CD8+ T cells against transduced hepatocytes was not inhibited by the CD4+ T cell fraction of PBMC expanded separately in vitro with Tregitopes only. We conclude that the use of Tregitopes represents a promising strategy for antigen-specific, Treg-mediated modulation of capsid-specific T cell responses. Disclosures: Martin: EpiVax: Employment. De Groot:EpiVax, Inc.: Employment, Equity Ownership.

Description: Adeno-associated virus (AAV)–mediated gene transfer of factor IX (F.IX) to the liver results in long-term expression of transgene in experimental animals, but only short-term expression in humans. Loss of F.IX expression is likely due to a cytotoxic immune response to the AAV capsid, which results in clearance of transduced hepatocytes. We used a nonhuman primate model to assess the safety of AAV gene transfer coupled with an anti–T-cell regimen designed to block this immune response. Administration of a 3-drug regimen consisting of mycophenolate mofetil (MMF), sirolimus, and the anti–IL-2 receptor antibody daclizumab consistently resulted in formation of inhibitory antibodies to human F.IX following hepatic artery administration of an AAV-hF.IX vector, whereas a 2-drug regimen consisting only of MMF and sirolimus did not. Administration of daclizumab was accompanied by a dramatic drop in the population of CD4+CD25+FoxP3+ regulatory T cells (Tregs). We conclude that choice of immunosuppression (IS) regimen can modulate immune responses to the transgene product upon hepatic gene transfer in subjects not fully tolerant; and that induction of transgene tolerance may depend on a population of antigen-specific Tregs.

Description: Abstract 3769 Recent advances in adeno-associated viral (AAV) vector-mediated gene transfer continue to offer hope for the correction of monogenic disorders such as hemophilia B. However, unanticipated T cell responses directed against viral capsid epitopes may limit the efficacy of AAV gene transfer. A phase I clinical study in which an AAV2 vector expressing human factor IX (FIX) was delivered systemically provided the first evidence that AAV vector administration at high doses may trigger the expansion of memory CD8+ T cells directed against AAV capsid epitopes. This response was associated with the loss of FIX transgene expression and a transient increase in liver enzymes. Additional studies in human subjects undergoing AAV gene transfer suggest that the capsid antigen load is an important determinant of capsid-specific T cell activation. Thus, strategies for the modulation of capsid T cell responses could contribute to achieving sustained transgene expression following high dose delivery of AAV. MHC class II peptide ligands identified within the human IgG Fc fragment (Tregitopes, Blood 2008;112:3303) have been shown to expand regulatory T cells (Tregs). Restimulation of human peripheral blood mononuclear cells (PBMC) in vitro with AAV capsid antigen in the presence of Tregitopes resulted in the suppression of capsid-specific CD8+ T cells and in the expansion of CD4+CD25+FoxP3+ Tregs. To better define the nature of Tregitope-induced Tregs, we depleted CD25+ cells from PBMC prior to in vitro restimulation. This completely prevented capsid-specific CTL suppression and the expansion of Tregs, suggesting that Tregitopes act by expanding natural Tregs. Cytokine ELISA on conditioned media from PBMC co-cultured with AAV antigen and Tregitopes showed a 50% decrease in IL-2 levels and a 〉500-fold increase in IL-10 levels. These results suggest that the effect of Tregitopes may be cytokine mediated. To test this hypothesis, we used a transwell system in which the CD4+ T cell fraction of Tregitope-restimulated PBMC was co-cultured with the capsid-specific CD8+ T cells. Without cell contact, a nearly 50% suppression of anti-capsid CD8+ T cell responses was observed. Further evidence supporting the role of cytokine-mediated suppression came from the observation that Tregitope-treated capsid-specific CD8+ T cells appeared to be anergic, and depletion of CD4+ T cells (Tregs) followed by a 24-hour incubation of CD8+CD4− T cells with IL-2 restored 〉80% of CTL activity. Finally, antigen specificity of Tregitope-induced Tregs was tested by expanding PBMC in vitro with HLA-B*0702-restricted epitopes from either the AAV capsid or the Epstein-Barr Virus (EBV). After in vitro restimulation, negatively-isolated CD4+ T cells expanded in the presence of EBV antigen and Tregitopes were co-incubated with either CD8+ T cells expanded against the AAV capsid or against EBV. Suppression of CTL activity was observed only when EBV Tregs were co-incubated with EBV CD8+ T cells. Similarly, Tregs isolated from AAV and Tregitope cultures suppressed AAV-specific CD8+ T cells but not EBV-specific CD8+T cells. These results suggest that inhibition of CD8+ T cell responses is antigen-specific. We conclude that Tregitopes induce the expansion of Tregs, which can mediate a potent antigen-specific inhibition of CD8+ T cell responses directed to the AAV capsid. Disclosures: Hui: Children's Hospital of Philadelphia: Patents & Royalties. Martin:EpiVax: Employment, Equity Ownership, Patents & Royalties. DeGroot:EpiVax: Employment, Equity Ownership. High:Children's Hospital of Philadelphia: Patents & Royalties. Mingozzi:Children's Hospital of Philadelphia: Patents & Royalties.

Description: Abstract 2050 Adeno-associated viral (AAV) vector-mediated gene transfer has shown great potential as a therapeutic platform for inherited and metabolic diseases. Systemic delivery of AAV vectors through the bloodstream is a safe, non-invasive, and potentially effective strategy to target a variety of organs, including liver, muscle, and brain. However, neutralizing antibodies (NAb) to AAV, highly prevalent in humans, constitute a major obstacle to successful gene transfer, particularly when a vector is delivered through the vasculature. Thus far, the liver was targeted to express the coagulation factor IX (F.IX) transgene in two clinical studies. In one study, a single-stranded AAV2 vector expressing the F.IX transgene was delivered through the hepatic artery to severe hemophilia B subjects at doses of 8×1010, 4×1011, and 2×1012 vector genomes (vg)/kg. Efficacy was observed in one subject from the high-dose cohort, who achieved peak F.IX transgene plasma levels of ∼10% of normal. The subjects infused at lower doses did not show any evidence of transgene expression, despite the fact that they did not have detectable NAb to AAV. In a second study, a self-complementary AAV8 vector expressing the F.IX transgene was delivered through peripheral vein infusion to severe hemophilia B subjects at doses similar to those administered in the AAV2 study, 2×1011, 6×1011, and 2×1012 vg/kg. All subjects enrolled in the AAV8 trial had evidence of transgene expression above baseline levels, despite the fact that some of the subjects had low-but-detectable anti-AAV8 NAb. Peak F.IX plasma levels at the high vector dose were 8–12% of normal, similar to the high dose of the AAV2 trial, suggesting that the vectors used in the two studies had comparable potency. Importantly, the vectors used in the two studies differed in empty capsid content, as the AAV2 vector preparation was essentially empty capsid-free and the AAV8 vector contained a 5–10 fold excess of empty capsids. The current study was undertaken to explore the role of empty capsids as a factor in the difference in outcome in the low- and mid- dose cohorts of the two trials. Our underlying hypothesis was that the presence of an excess of empty capsids effectively absorbs low-level neutralizing and non-neutralizing antibodies, and permits transduction even in their presence. Using a newly developed AAV antibody dot-blot assay, we demonstrate that adult human subjects with a low to undetectable NAb titer (1:1) as assessed by a commonly used assay do, in fact, carry significant amounts of anti-AAV antibodies. Conversely, children aged one year appear to be truly naïve for anti-AAV humoral immunity. Using C57BL/6 mice passively immunized with purified human IgG injected intraperitoneally 24 hours before vector administration, we further demonstrate that the same low levels of anti-AAV antibodies found in humans (NAb titer of 1:1–1:3) can block 〉90% of liver transduction after peripheral vein delivery of AAV8 vectors expressing F.IX at doses of 1×1012 vg/kg, comparable to those tested in the clinic. We next demonstrated that the inhibitory effect of low titer (1:1–1:3) anti-AAV antibodies can be overcome by adding a 5 to 10-fold excess of empty capsids to the final formulation of AAV8 vector, and that empty capsid content can be carefully titrated as a function of the animal's anti-AAV NAb in order to achieve efficient target organ transduction, even at titers 〉1:100. However, the beneficial effect of empty capsids on liver transduction is lost when a 1000-fold excess of AAV8 empty capsids are added to the formulation of AAV8 vectors, due to receptor binding competition. This inhibitory effect could be avoided by using AAV2 empty capsids, which efficiently protect AAV8 vectors from NAb without inhibiting transduction. These results were confirmed in non-human primates, a natural host for AAV8, in which a 5 to 6-fold increase in liver transduction was achieved by formulating vector in 5–10 fold excess AAV8 empty capsids, reaching levels of F.IX expression of 10 to 20% of normal. Application of these findings to the development of personalized formulations of vector product for intravascular delivery will facilitate safe, effective AAV-mediated gene transfer in settings in which vectors are delivered through the systemic circulation. Disclosures: Mingozzi: Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Anguela:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Wright:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. High:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties.

Description: Abstract 752 Introduction: We are conducting a phase I/II clinical trial of factor IX gene transfer for severe hemophilia B. In the trial we are using a serotype-8 pseudotyped self-complementary adeno-associated virus (scAAV) vector expressing a codon-optimized coagulation factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco). We have previously reported the early safety and efficacy of our novel gene transfer approach in six patients with severe hemophilia B following a single peripheral vein infusion of one of three vector doses (low [2×1011 vector particles (vp)/kilogram weight (kg)], intermediate [6×1011 vp/kg], or high dose [2×1012 vp/kg]) (Nathwani et al, NEJM 365:2357–65, 2011). AAV-mediated expression of FIX at 1–6% of normal was established in all six participants with an initial follow-up of between 6–14 months following gene transfer. We now report longer follow-up of these participants, as well as data from two additional participants recently enrolled at the high dose level. Methods: We have now infused scAAV2/8-LP1-hFIXco in eight subjects with severe hemophilia B (FIX activity,

Description: Abstract 5 Background: Hemophilia B (HB), an X-linked bleeding disorder, is ideally suited for gene therapy. We investigated a novel approach using peripheral vein infusion of a single dose of a serotype-8 pseudotyped self-complementary adeno-associated virus (AAV) vector expressing a codon-optimized coagulation factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco). Methods: Six severe HB subjects (FIX ≤1%) were enrolled sequentially into one of three dose cohorts with two subjects in each group. Vector was administered without immunosuppression. The subjects were followed for 6–16 months post treatment. Results: AAV-mediated expression of FIX at 2–11% of normal was observed in all subjects. Four of the six have discontinued prophylaxis and remain free of spontaneous hemorrhage. The other two have increased the interval between FIX prophylaxes. A high-dose subject developed asymptomatic, transient elevation of serum transaminases associated with detection of AAV8 capsid specific T cells in peripheral blood. The second high-dose subject experienced a slight increase of liver enzymes, of less clear etiology. Treatment of each with a short course of steroids led to rapid normalization of the transaminases and maintenance of FIX levels in the 3–11% range. Conclusion: Peripheral vein administration of scAAV2/8-LP1-hFIXco was well tolerated and resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype. Immune-mediated clearance of AAV-transduced hepatocytes remains a concern but our data suggest that this process may be controlled with a short course of steroids without loss of transgene expression. Hence, our novel approach shows promise for gene therapy of HB and other protein deficiencies. (ClinicalTrials.gov number, NCT00979238) Disclosures: Nathwani: Amsterdam Molecular Therapeutics: Patents & Royalties. Gray:Amsterdam Molecular Therapeutics: Patents & Royalties. Davidoff:Amsterdam Molecular Therapeutics: Patents & Royalties.

Description: In a clinical study of gene transfer for hemophilia B an adeno-associated virus vector serotype 8 (AAV8) expressing a self-complementary liver-specific expression cassette for the factor IX (FIX) transgene was administered intravenously in ten affected subjects. The results of the first part of the study have been published (NEJM 365:2357-65, 2011). In this abstract we present the immunomonitoring data, using Interferon-gamma (IFN-γ) ELISpot and polyfunctional T cell analysis of peripheral blood mononuclear cells (PBMCs) to monitor cellular immune responses to vector capsid and to Factor IX. We have previously shown that the cellular immune response was directed solely towards AAV capsid epitopes, not FIX, and that the response was dose-dependent. Out of six subjects infused in the high dose cohort (2x1012vg/kg), 4/6 manifested a minor rise in liver enzyme levels and detection of capsid-specific T cell reactivitiy in the ELISpot assay at ∼7-10 weeks post vector infusion. Maximum results on IFN- γ ELISpots ranged from 200-500 sfu/million cells. In two of these cases a modest decline in FIX level also occurred. Prompt initiation of prednisolone reversed these effects and rescued FIX levels. The remaining two subjects infused at the high dose, showed no rise in liver enzyme levels at any time point. However capsid reactive T cells were detectable in one subject as early as one to two weeks after vector infusion in peripheral blood by IFN-γ ELISpot assay, while no activation at all was detected in the other subject, possibly due to low cell recovery and viability of the cells. A similar immune response profile, with early detection of activated T cells but no rise in liver enzymes, was also observed in both subjects in the intermediate dose cohort in the first part of this study. Polyfunctional T cell analysis revealed concurrent Interleukin-2, Tumor necrosis factor-alpha and CD107a positivity in activated T cells at the peak of activation. Furthermore it showed that capsid-specific early T cell responses were detectable in the CD4+ T cell and later in the CD8+T cell compartment. Long-term immune monitoring of all subjects is ongoing. Importantly in one of the first two subjects treated at the high dose, capsid reactive T cells were detected by ELISpot 1.5 years after gene transfer; these cells were not detected in the other subject in whom long-term follow-up samples are available. Of note, capsid-reactive T cells were also seen at late time points (〉1 year after infusion) in a middle dose subject and a low dose subject. Despite detectable T cell reactivity towards the AAV capsid in the peripheral blood FIX expression remained stable, suggesting that there is a short window of time during which transduced hepatocytes present a target for cytotoxic T cells, and that T cell positivity after this window is without any clinical consequences. In conclusion, for this scAAV8 vector there appears to be a critical threshold vector dose for a clinically detectable immune response, starting at 2x1012 vg/kg. The clinically detectable response occurred in four out of six subjects so far, and was manifest within a critical time interval of 7-10 weeks post infusion. The capsid-specific response was polyfunctional and detected in CD4+ and CD8+T cells in peripheral blood. It is important to note that not all subjects treated at the high dose developed an immune response. However, given the limited dataset, it is not yet possible to define predictive parameters, e.g. HLA type of a subject, for an immune response. Continued monitoring and future studies with more subjects will be necessary to confirm the presented findings, in particular time and rate of occurrence of a cellular response as well as successful treatment with a short course of Prednisolon. Disclosures: Tuddenham: Pfizer: Consultancy. Reiss:Hemophilia of Georgia: Honoraria. High:BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

Description: Key Points Crosspriming of AAV capsid-specific CD8+ T cells requires cooperation between distinct subsets of DCs. Innate immune sensing of the viral DNA genome induces cross-presentation of viral capsid in trans.

Description: Key Points A murine model was developed for capsid-specific CD8 cell responses in AAV gene therapy for hemophilia. Y-F mutant capsid minimizes the effect of anticapsid CD8+ T cells on hepatocyte-derived factor IX expression in mice and in human cells.

Description: In a clinical trial for adeno-associated virus serotype 1 (AAV-1)–mediated gene transfer to muscle for lipoprotein lipase (LPL) deficiency, 1 subject from the high-dose cohort experienced a transient increase in the muscle enzyme creatine phosphokinase (CPK) 4 weeks after gene transfer. Simultaneously, after an initial downward trend consistent with expression of LPL, plasma triglyceride levels returned to baseline. We characterized B- and T-cell responses to the vector and the transgene product in the subjects enrolled in this study. IFN-γ enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining assays performed on peripheral blood mononuclear cells (PBMCs) from the subject who experienced the CPK elevation showed the activation of capsid-specific CD4+ and CD8+ T cells. Four of 8 subjects had detectable T-cell responses to capsid with dose-dependent kinetics of appearance. Subjects with detectable T-cell responses to capsid also had higher anti–AAV-1 IgG3 antibody titer. No subject developed B- or T-cell responses to the LPL transgene product. These findings suggest that T-cell responses directed to the AAV-1 capsid are dose-dependent. Whether they also limit the duration of expression of the transgene at higher doses is unclear, and will require additional analyses at later time points.